This application is a 371 of PCT/EP99/00646, Feb. 2, 1999.
The invention relates to sulfonylamino acid and sulfonylamino hydroxamic acid derivatives and to processes for their preparation, pharmaceutical compositions comprising said compounds, a method of inhibiting matrix-degrading metalloproteinases in mammals using such compounds and the use of these derivatives as medicaments.
The present invention relates to sulfonylamino acid and sulfonylamino hydroxamic acid derivatives of formula I 
in which
W is xe2x80x94OH or xe2x80x94NHOH;
X is
a) an unsubstituted or substituted heterocyclic radical, selected from the group consisting of pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, 4-piperidinyl, pyridyl, pyrazinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane, tetrahydro-1,1-dioxothienyl, benzothiazolyl, benzoxazolyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl, dihydrobenzoisothiazolyl, dihydroquinazolinyl, tetrahydroquinazolinyl and 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one carbon atom-containing ring, in which each ring of the heterocyclic radical containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms;
with the proviso that when X is a nitrogen containing heterocyclic radical, the heterocyclic radical is attached to the (CH2)m moiety by a ring nitrogen and the proviso that nitrogen and sulfur heteroatoms of the heterocyclic radical may also be oxidized;
b) xe2x80x94NR1SO2R2, in which
R1 is hydrogen, alkyl, heterocyclylalkyl, aralkyl or heteroarylalkyl and
R2 is hydrogen, alkyl, heterocyclylalkyl, aralkyl, heteroarylalkyl, aryl or heteroaryl;
c) heterocyclylalkylthio;
d) xe2x80x94CONR2R3, in which
R2 and R3 taken together with the nitrogen atom to which they are attached form a 5- to 7- membered ring, which may optionally contain another heteroatom selected from oxygen, nitrogen and sulfur; or
e) xe2x80x94NR1COR2, in which
R1 is hydrogen, alkyl, heterocyclylalkyl, aralkyl or heteroarylalkyl and
R2 is hydrogen, heterocyclylalkyl, aralkyl, heteroar ylalkyl or aryl;
Y is carbon, nitrogen, oxygen or sulfur, provided that when Y is carbon, n is 2;
Z is alkyl, aryl, alkoxy, aryloxy, aralkoxyaryl, aralkoxyheteroaryl, heteroaryl, heterocyclyl, heteroaryloxy, xe2x80x94CONR2R3, xe2x80x94NR1COR2, xe2x80x94NR1CONR2R3, xe2x80x94OCONR2R3, xe2x80x94NR1COOR4, or xe2x80x94SO2R2, in which
R1 is hydrogen, alkyl, heterocyclylalkyl, aralkyl or heteroarylalkyl and
R2 and R3 are independently hydrogen, alkyl, heterocyclylalkyl, aralkyl, heteroarylalkyl, aryl or heteroaryl; or
R2 and R3 taken together with the nitrogen atom to which they are attached form a 5- to 7- membered ring, which may optionally contain another heteroatom selected from oxygen, nitrogen and sulfur;
R4 is alkyl, heterocyclylalkyl, aralkyl, aryl or heteroaryl;
m represents an integer from one to six; and
n represents the integer one or two;
and to pharmaceutically acceptable salts thereof.
Compounds of formula I are inhibitors of matrix-degrading metalloproteinases and are useful for the treatment of conditions related thereto.
The present invention provides for compounds of formula I, pharmaceutical compositions employing such compounds and for methods of using such compounds. Listed below are definitions of various terms used to describe the compounds of the instant invention. These definitions apply to the terms as they are used throughout the specification (unless they are otherwise limited in specific instances either individually or as part of a larger group).
The term xe2x80x9cheterocyclic rad icalxe2x80x9d refer s to an optionally substituted, fully saturated or unsaturated, aro matic or nonaromatic cyclic group, for example, which is a 4 t o 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring system, which has at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic radicals containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms, where the nitrogen and sulfur heteroatoms may also optionally be oxidized.
Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, isoxazolyl, isoxazolinyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, thienyl, tetrahydrofuryl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl , azepinyl, 4-piperidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothienyl, and the like.
Exemplary bicyclic heterocyclic groups include indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), pyrrolopyridyl, dihydrobenzoisothiazolyl, dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), tetrahydroquinazolinyl and the like.
Exemplary tricyclic heterocyclic groups include tetrahydroimidazo[1,5-b]isoquinolinyl, carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like.
The term xe2x80x9cheterocyclic radicalxe2x80x9d also includes substituted heterocyclic groups. Substituted heterocyclic groups refer to heterocyclic groups substituted with 1, 2, 3, 4 or 5 of the following:
(a) alkyl;
(b) hydroxy (or protected hydroxy);
(c) halo;
(d) oxo (i.e.=O);
(e) amino, alkylamino or dialkylamino;
(f) alkoxy;
(g) cycloalkyl;
(h) carboxy;
(i) heterocyclyloxy;
(j) alkoxycarbonyl, such as unsubstituted lower alkoxycarbonyl;
(k) carbamyl, alkylcarbamyl or dialkylcarbamyl;
(l) mercapto;
(m) nitro;
(n) cyano;
(o) sulfonamido, aminosulfonyl, alkyl or dialkylsulfonyl;
(p) aryl or heteroaryl;
(q) alkylcarbonyloxy;
(r) arylcarbonyloxy;
(s) arylthio;
(t) aryloxy;
(u) alkylthio;
(v) formyl;
(w) arylalkyl; or
(x) aryl substituted with alkyl, cycloalkyl, alkoxy, hydroxy, amino, alkylamino, dialkylamino or halo.
The term xe2x80x9calkylxe2x80x9d refers to optionally substituted straight or branched chain hydrocarbon groups having 1 to 8 carbon atoms, preferably 1 to 5 carbons. Exemplary unsubstituted alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, the various branched chain isomers thereof, such as isopropyl, t-butyl, isobutyl, isohexyl, 4,4-dimethylpentyl, 2,2,4-trimethylpentyl and the like. Substituted alkyl groups include said alkyl groups substituted by one or more substituents selected from halogen, alkoxy, cycloalkyl, hydroxy, amino, nitro, cyano or thiol.
The term xe2x80x9calkoxyxe2x80x9d refers to any of the above alkyl groups linked to an oxygen atom.
The term xe2x80x9ccycloalkylxe2x80x9d refers to saturated cyclic hydrocarbon groups containing 3 to 7 ring carbons with cyclopropyl, cyclopentyl and cyclohexyl being preferred.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to chlorine, bromine, iodine and fluorine.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring portion, such as phenyl, naphthyl, tetrahydronaphthyl, or biphenyl groups, each of which may optionally be substituted by one to four substituents such as alkyl, halo, hydroxy, alkoxy, amino, thiol, nitro, cyano, carboxy, heterocycle and the like.
The term xe2x80x9caralkylxe2x80x9d refers to an aryl group bonded directly through an alkyl group, such as benzyl.
The term xe2x80x9caryloxyxe2x80x9d refers to an aryl group linked to an oxygen atom.
The term xe2x80x9caralkoxyxe2x80x9d refers to an aryl group bonded directly through an alkoxy group.
The term xe2x80x9caroylxe2x80x9d refers to aryl-C(O)xe2x80x94.
The term xe2x80x9cheterocyclylxe2x80x9d denotes a heterocyclic radical.
The term xe2x80x9cheterocyclylalkylxe2x80x9d denotes a heterocyclic radical bonded directly through an alkyl group.
The term xe2x80x9cheterocyclyloxyxe2x80x9d denotes a heterocyclic group bonded through an oxygen bridge.
The term xe2x80x9cheteroarylxe2x80x9d refers to an aromatic heterocyclic radical.
The term xe2x80x9cheteroarylalkylxe2x80x9d refers to an aromatic heterocyclic radical bonded directly through an alkyl group.
The term xe2x80x9cheteroarylsulfonylxe2x80x9d refers to heteroaryl-S(O)2xe2x80x94.
The term xe2x80x9cheteroaroylxe2x80x9d refers to heteroaryl-C(O)xe2x80x94.
Throughout the specification, groups and substituents thereof are chosen to provide stable moieties and compounds.
The compounds of the invention depending on the nature of the substituents, possess one or more asymmetric carbon atoms. The resulting diastereoisomers, enantiomers and geometric isomers are encompassed by the instant invention.
Preferred are the compounds of the invention wherein the configuration of the asymmetric carbon atom of compounds of formula I is assigned the (R)-configuration.
Further preferred are the compounds of formula I in which W is xe2x80x94OH or xe2x80x94NHOH; X is an unsubstituted or substituted heterocyclic radical, selected from the group consisting of pyrrolidinyl, oxetanyl, pyrazolinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolinyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, 4-piperidinyl, pyrazinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, 1,3-dioxolane, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, tetrahydro-isoquinolinyl, tetrahydro-1,1-dioxothienyl, quinuclidinyl, quinolinyl, isoquinolinyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl, dihydro-benzoisothiazolyl, dihydroquinazolinyl, tetrahydroquinazolinyl and 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one carbon atom-containing ring, in which each ring of the heterocyclic radical containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms; and the remaining symbols and radicals have the same meaning as given above.
Another preferred embodiment of the invention is a compound of formula I in which W is xe2x80x94OH or xe2x80x94NHOH; X is an unsubstituted or substituted heterocyclic radical, selected from the group consisting of pyrazolyl, oxetanyl, pyrazolinyl, imidazolinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, oxadiazolyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxoazepinyl, pyridyl, pyrazinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane, tetrahydro-1,1-dioxothienyl, benzothiazolyl, benzoxazolyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzopyranyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl, dihydrobenzoisothiazolyl, dihydroquinazolinyl, tetrahydroquinazolinyl and 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one carbon atom-containing ring, in which each ring of the heterocyclic radical containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms; and the remaining symbols and radicals have the same meaning as given above.
Further preferred are the compounds of formula I in which W is xe2x80x94OH or xe2x80x94NHOH; X is a nitrogen containing heterocyclic radical; Y is carbon and n is two; Z is aryl, aryloxy, heteroaryl or heteroaryloxy and m represents an integer from two to four.
Particularly preferred compounds of the formula I are those in which W is xe2x80x94OH or xe2x80x94NHOH; X is one of the following: 1,2,3,4-tetrahydro-1-methyl-2,4-dioxo-quinazolinyl; 3,4,4-trimethyl-2,5-dioxoimidazolinyl; 4-methylbenzenesulfonylamino; or 1,1,3-trioxo-2,3-dihydrobenzoisothiazolyl; Z is aryl, aryloxy, heteroaryl or heteroaryloxy; Y is carbon, nitrogen, oxygen or sulfur, provided that when Y is carbon, n is 2; n represents the integer one or two and m represents an integer from two to four.
Particularly preferred compounds of the formula I are those in which W is xe2x80x94OH and the remaining symbols and radicals have the same meaning as given above.
Another preferred embodiment of the invention is a compound of formula I in which W is xe2x80x94OH or xe2x80x94NHOH; X is xe2x80x94CONR2R3, in which R2 and R3 taken together with the nitrogen atom to which they are attached form a 5- to 7- membered ring, which contains oxygen as another heteroatom; Y is carbon; n is two; Z is aryl or aryloxy and m represents an integer from one to three.
Also preferred is a compound of formula I in which W is xe2x80x94OH; X is 1,2,3,4-tetrahydro-1-methyl-2,4-dioxo-quinazolinyl or 1,1,3-trioxo-2,3-dihydrobenzo-isothiazolyl; Y is carbon; n is two; Z is aryl or aryloxy, whereby in each case aryl is unsubstituted or substituted by halogen and m represents an integer from two to four.
Another preferred embodiment of the invention is a compound of formula I in which W is xe2x80x94OH or xe2x80x94NHOH; X is xe2x80x94NR1COR2, in which R1 is hydrogen and R2 is aralkyl or aryl; Y is carbon; n is two; Z is alkoxy or aryl; and m represents an integer from three to four. Also preferred is a compound of formula I in which W is xe2x80x94OH or xe2x80x94NHOH; X is xe2x80x94NR1SO2R2, in which R1 is hydrogen and R2 is alkyl, heterocyclylalkyl, aralkyl, heteroarylalkyl, aryl or heteroaryl; Y is carbon; n is two; Z is alkoxy or aryl; and m represents an integer from three to four.
Pharmaceutically acceptable salts of any acidic compounds of the invention are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethylammonium, diethylammonium, and tris-(hydroxymethyl)-methylammonium salts.
Similarly acid addition salts, such as of mineral acids, organic carboxylic, and organic sulfonic acids e.g. hydrochloric acid, methanesulfonic acid, maleic acid, are possible provided a basic group, such as pyridyl, constitutes part of the structure.
Compounds of formula I where X is a heterocyclic radical or heterocyclylalkylthio may be prepared from N-(diphenylmethylene)glycine t-butyl ester (commercially available) and compounds of formula
Yxe2x80x94(CH2)mxe2x80x94Xxe2x80x83xe2x80x83(II)
where Y is a leaving group such as a halide such as chloride, bromide or iodide; or a sulfonate such as methenesulfonate, trifluoromethanesulfonate or methylbenzenesulfonate (prepared as described in the literature); by treatment with a base such as sodium or potassium hydride in a organic solvent such as tetrahydrofuran and N,N-dimethylformamide to form compounds of formula III. 
Compounds of formula III can be converted to compounds of formula IV 
by treatment with a mild acid such as 4-methylbenzenesulfonic acid in a organic solvent such as acetonitrile and tetrahydrofuran in a presence of a small amount of water.
Sulfonylation of compounds of formula IV with sulfonyl chlorides such as 4xe2x80x2-chlorobiphenyl-4-sulfonyl chloride or a compound of formula V 
produces compounds of formula VI. 
The sulfonylation can be carried out in the presence of a base such as triethylamine or N-methylmorpholine in a organic solvent such as dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide. Sulfonyichlorides of formula V can be obtained as described in the art.
Compounds of formula VI can be converted to compounds of formula I where W is a hydroxyl group by treatment with anhydrous acid such as trifluoroacetic acid or hydrochloric acid in a organic solvent such as dichloromethane, diethyl ether or ethyl acetate. The reaction can be carried out without solvent when trifluoroacetic acid is used.
Compounds of formula I where W is xe2x80x94NHOH can be obtained from compounds of formula I where W is a hydroxyl group via a reaction with protected hydroxylamines such as trityl-, allyl- or t-butylhydroxylamine. The reactions can be carried out in the presence of a coupling agent such as 1-hydroxy-7-azabenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and a base such as triethylamine or N-methylmopholine in a organic solvent such as dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide. The protecting groups can be removed as described in the art.
Alternatively, compounds of formula I where X is a heterocyclic radical or heterocyclylalkylthio and m is 2 or 3 can be obtained from compounds of formula VII 
where mxe2x80x2 is an integer of 2 or 3 and Ra, Rb and Rc are independently suitable protecting groups. For example, when Ra is methyl or ethyl and Rb and Rc are e.g. t-butyl, compounds of formula VII can be treated with a base such as lithium hydroxide, sodium hydroxide or potassium hydroxide in an aqueous solution of a organic solvent such as tetrahydrofuran, dioxane, methanol or ethanol to form compounds of formula VIII 
Compounds of formula VIII are then treated with alkyl chloroformate such as ethylchloroformate or isobutylchloroformate in a presence of a base such as N-methylmorpholine or triethylamine in organic solvents such as tetrahydrofuran, dioxane or ethylene glycol dimethyl ether followed by reduction using sodium borohydride to provide compounds of formula IX. 
Compounds of formula IX are converted to compounds of formula X 
where Ya is a leaving group such as a halide, such as iodide, bromide or chloride or a sulfonate such as methanesulfonate, trifluoromethanesulfonate or 4-methylbenzenesulfonate using standard literature procedures.
Compounds of formula X are then treated with a compound (Xxe2x80x2), where Xxe2x80x2 is a heterocyclic radical such as saccharin, 1,2,3,4-tetrahydro-1-methyl-2,4-dioxoquinazoline, 3,4,4-trimethyl-2,5-dioxoimidazolidine or heterocyclylalkylthiol; in the presence of a base such as sodium hydride, potassium hydride, potassium carbonate or caesium carbonate in an organic solvent such as N,N-dimethylformamide to form compounds of formula Xl. 
Compounds of formula XI are then selectively deprotected at the nitrogen atom using procedures known in the art to form compounds of formula XII. 
For example, where Rb and Rc are t-butyl, deprotection can be carried out by treatment with an anhydrous acid such as trifluoroacetic acid in an organic solvent such as dichloromethane.
Compounds of formula XII are then sulfonylated with sulfonyl chlorides of formula V such as 4xe2x80x2-chlorobiphenyl-4-sulfonyl chloride. The sulfonylation can be carried out using a base such as triethylamine or N-methylmorpholine in an organic solvent such as dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide to form compounds of formula XIII. 
Compounds of formula XIII are converted to compounds of formula IA 
where W is OH by removal of the carboxyl protecting group using known methods. For example, where Rc is a t-butyl group, deprotection can be carried out by treatment with an anhydrous acid such as trifluoroacetic acid or hydrochloric acid in an organic solvent such as dichloromethane, diethyl ether or ethyl acetate. The reactions can be carried out without solvent when trifluoroacetic acid is used.
Compounds of formula IA where W is xe2x80x94NHOH can be obtained from compounds of formula IA where W is a hydroxyl group via a reaction with protected hydroxylamines such as trityl-, allyl- or t-butylhydroxylamine. The reactions can be carried out in the presence of a coupling agent such as 1-hydroxy-7-azabenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and a base such as triethylamine or N-methylmopholine in an organic solvent such as dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide. The protecting groups can be removed as described in the art.
Compounds of formula VII can be prepared by methods disclosed in the literature (Schoenfelder, A.: Mann, A., Synth. Commun., 20, 2585-8 (1990)).
Compounds of formula I where m equals 1 or 2 and X is xe2x80x94CONR2R3, can be prepared from compounds of formula VIII using procedures known in the art or by modification of the procedures described herein.
Compounds of formula I when m equals 3 or 4 and X is xe2x80x94NHxe2x80x94SO2xe2x80x94R2 or NHCOR2 can be prepared from compounds of formula XV 
where mxe2x80x3 is an integer of 3 or 4 (commercially available or known in the literature), using procedures known in the art or by modification of the procedures described herein. Compounds of formula I where m equals 1 and X is heterocyclylalkylthio can also be prepared from compounds of formula XVI 
where Yb is a protecting group such as an acyl or can form a disulfide dimer, (commercially available or known in the literature), using procedures known in the art or by modification of the procedures described herein.
Other compounds of formula I may be prepared by modification of the procedures and examples described herein.
The starting compounds and intermediates which are converted to the compounds of the invention in a manner described herein, functional groups present, such as amino, thiol, carboxyl, and hydroxy groups, are optionally protected by conventional protecting groups that are common in preparative organic chemistry. Protected amino, thiol, carboxyl, and hydroxy groups are those that can be converted under mild conditions into free amino, thiol, carboxyl and hydroxy groups without the molecular framework being destroyed or other undesired side reactions taking place.
The purpose of introducing protecting groups is to protect the functional groups from undesired reactions with reaction components under the conditions used for carrying out a desired chemical transformation. The need and choice of protecting groups for a particular reaction is known to those skilled in the art and depends on the nature of the functional group to be protected (hydroxy group, amino group, etc.), the structure and stability of the molecule of which the substituent is a part and the reaction conditions.
Well-known protecting groups that meet these conditions and their introduction and removal are described, for example, in J. F. W. McOmie, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, London, New York, 1973, T. W. Greene, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, Wiley, N.Y., 1991.
In the processes cited herein, reactive functional derivatives of carboxylic acids represent, for example, anhydrides (especially mixed anhydrides), acid halides, acid azides, lower alkyl esters, and activated esters thereof. Mixed anhydrides are preferably such from pivalic acid, or a lower alkyl (ethyl, isobutyl) hemiester of carbonic acid; acid halides are for example chlorides or bromides; activated esters for example succinimido, phthalimido or 4-nitrophenyl esters; lower alkyl esters are for example the methyl or ethyl esters.
The above-mentioned reactions are carried out according to standard methods, in the presence or absence of diluent, preferably such as are inert to the reagents and are solvents thereof, of catalysts, condensing or said other agents respectively and/or inert atmospheres, at low temperatures, room temperature or elevated temperatures (preferably at or near the boiling point of the solvents used), and at atmospheric or super-atmospheric pressure. The preferred solvents, catalysts and reaction conditions are set forth in the appended illustrative Examples.
The invention further includes any variant of the present processes, in which an intermediate product obtainable at any stage thereof is used as starting material and the remaining steps are carried out, or in which the starting materials are formed in situ under the reaction conditions, or in which the reaction components are used in the form of their salts or optically pure antipodes.
Compounds of the invention and intermediates can also be converted into each other according to methods generally known per se.
The invention also relates to any novel starting materials and processes for their manufacture.
Depending on the choice of starting materials and methods, the new compounds may be in the form of one of the possible isomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) isomers, optical isomers (antipodes), racemates, or mixtures thereof. The aforesaid possible isomers or mixtures thereof are within the purview of this invention.
Any resulting mixtures of isomers can be separated on the basis of the physico-chemical differences of the constituents, into the pure geometric or optical isomers, diastereoisomers, racemates, for example by chromatography and/or fractional crystallization.
Any resulting racemates of final products or intermediates can be resolved into the optical antipodes by known methods, e.g. by separation of the diastereoisomeric salts thereof, obtained with an optically active acid or base, and liberating the optically active acidic or basic compound. The carboxylic acid intermediates can thus be resolved into their optical antipodes e.g. by fractional crystallization of d- or l-(alpha-methylbenzylamine, cinchonidine, cinchonine, quinine, quinidine, ephedrine, dehydroabietylamine, brucine or strychnine)-salts. Racemic products can also be resolved by chiral chromatography, e.g. high pressure liquid chromatography using a chiral adsorbent.
Finally, compounds of the invention are either obtained in the free form, or as a salt thereof if salt forming groups are present.
Acidic compounds of the invention may be converted into salts with pharmaceutically acceptable bases, e.g. an aqueous alkali metal hydroxide, advantageously in the presence of an ethereal or alcoholic solvent, such as a lower alkanol. From the solutions of the latter, the salts may be precipitated with ethers, e.g. diethyl ether. Resulting salts may be converted into the free compounds by treatment with acids. These or other salts can also be used for purification of the compounds obtained.
Compounds of the invention having basic groups can be converted into acid addition salts, especially pharmaceutically acceptable salts. These are formed, for example, with inorganic acids, such as mineral acids, for example sulfuric acid, a phosphoric or hydrohalic acid, or with organic carboxylic acids, such as (C1-C4)-alkanecarboxylic acids which, for example, are unsubstituted or substituted by halogen, for example acetic acid, such as saturated or unsaturated dicarboxylic acids, for example oxalic, succinic, maleic or fumaric acid, such as hydroxycarboxylic acids, for example glycolic, lactic, malic, tartaric or citric acid, such as amino acids, for example aspartic or glutamic acid, or with organic sulfonic acids, such as (C1-C4)-alkylsulfonic acids (for example methanesulfonic acid) or aryisulfonic acids which are unsubstituted or substituted (for example by halogen). Preferred are salts formed with hydrochloric acid, methanesulfonic acid and maleic acid.
In view of the close relationship between the free compounds and the compounds in the form of their salts, whenever a compound is referred to in this context, a corresponding salt is also intended, provided such is possible or appropriate under the circumstances.
The compounds, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.
The pharmaceutical compositions according to the invention are those suitable for enteral, such as oral or rectal, transdermal and parenteral administration to mammals, including man, to inhibit matrix-degrading metalloproteinases, and for the treatment of disorders responsive thereto, comprising an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers.
The pharmacologically active compounds of the invention are useful in the manufacture of pharmaceutical compositions comprising an effective amount thereof in conjunction or admixture with excipients or carriers suitable for either enteral or parenteral application. Preferred are tablets and gelatin capsules comprising the active ingredient together with a) diluents, e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g. silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders e.g. magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g. starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbants, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
Suitable formulations for transdermal application include an effective amount of a compound of the invention with carrier. Advantageous carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. Characteristically, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
Suitable formulations for topical application, e.g. to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art.
The pharmaceutical formulations contain an effective matrix-degrading metalloproteinase inhibiting amount of a compound of the invention as defined above, either alone or in combination with another therapeutic agent, e.g. an anti-inflammatory agent with cyclooxygenase inhibiting activity, or other antirheumatic agents such as methotrexate, each at an effective therapeutic dose as reported in the art. Such therapeutic agents are well-known in the art.
Examples of antiinflammatory agents with cyclooxygenase inhibiting activity are diclofenac, naproxen, ibuprofen, and the like.
In conjunction with another active ingredient, a compound of the invention may be administered either simultaneously, before or after the other active ingredient, either separately by the same or different route of administration or together in the same pharmaceutical formulation.
The dosage of active compound administered is dependent on the species of warm-blooded animal (mammal), the body weight, age and individual condition, and on the form of administration. A unit dosage for oral administration to a mammal of about 50 to 70 kg may contain between about 10 and 1000 mg, advantageously between about 25 and 500 mg of the active ingredient.
The compounds of the invention inhibit matrix degrading metalloproteinase such as gelatinase, stromelysin, coliagenase, (including collagenase 1 and 3), and macrophage metalloelastase, and membrane type matrix metalloproteinases, such as MT-MMP 1 and 2. They are particularly useful as collagenase-3 inhibitors.
Thus the compounds of the invention inhibit matrix degradation and are useful for the treatment of gelatinase-, stromelysin-, collagenase-, and macrophage metalloelastase-dependent pathological conditions in mammals. Such conditions include malignant and non-malignant tumors (by inhibiting tumor growth, tumor metastasis, tumor progression or invasion and/or tumor angiogenesis), such tumors including e.g. breast, lung, bladder, colon, ovarian, and skin cancer. Other conditions to be treated with the compounds of the invention include rheumatoid arthritis osteoarthritis, bronchial disorders (such as asthma by inhibiting the degradation of elastin), atherosclerotic conditions (by e.g. inhibiting rupture of atherosclerotic plaques), as well as acute coronary syndrome, heart attacks (cardiac ischemia), strokes (cerebral ischemias), restenosis after angioplasty, and also vascular ulcerations, ectasia and aneurysms.
Further conditions to be treated with the compounds of the invention are inflammatory demyelinating disorders of the nervous system in which myelin destruction or loss is involved (such as multiple sclerosis), optic neuritis, neuromyelitis optica (Devic""s disease), diffuse and transitional sclerosis (Schilder""s disease) and acute disseminated encephalomyelitis, also demyelinating peripheral neuropathies such as Landry-Guillain-Barre-Strohl syndrome for motor defects; also tissue ulceration (e.g. epidermal and gastric ulceration), abnormal wound healing, periodental disease, bone disease (e.g. Paget""s disease and osteoporosis). Also endometriosis, septic shock, inflammatory bowel disease, Crohn""s disease and the like.
Ocular applications of the compounds of the invention include the treatment of ocular inflammation, corneal ulcerations, pterygium, keratitis, keratoconus, open angle glaucoma, retinopathies, and also their use in conjunction with refractive surgery (laser or incisional) to minimize adverse effects.
The compounds are particularly useful for the treatment of e.g. inflammatory conditions, osteoarthritis, rheumatoid arthritis and tumors.
Beneficial effects are evaluated in pharmacological tests generally known in the art, and as illustrated herein.
The above-cited properties are demonstrable in in vitro and in vivo tests, using advantageously mammals, e.g. rats, guinea pigs, dogs, rabbits, or isolated organs and tissues, as well as mammalian enzyme preparations. Said compounds can be applied in vitro in the form of solutions, e.g. preferably aqueous solutions, and in vivo either enterally or parenterally, advantageously orally, e.g. as a suspension or in aqueous solution. The dosage in vitro may range between about 10xe2x88x925 molar and 10xe2x88x9210 molar concentrations. The dosage in vivo may range, depending on the route of administration, between about 0.1 and 100 mg/kg.
Antiinflammatory activity can be determined in standard inflammation and arthritic animal models well-known in the art, e.g. the adjuvant arthritis model in rats and the collagen II induced arthritis model in mice (Mediators of Inflam. 1, 273-279 (1992).
One test to determine the inhibition of stromelysin activity is based on its hydrolysis of Substance P using a modified procedure of Harrison et al (Harrison, R. A., Teahan J., and Stein R., A semicontinuous, high performance chromatography based assay for stromelysin, Anal. Biochem. 180, 110-113 (1989)). In this assay, Substance P is hydrolyzed by recombinant human stromelysin to generate a fragment, Substance P 7-11, which can be quantitated by HPLC. In a typical assay, a 10 mM stock solution of a compound to be tested is diluted in the assay buffer to 50 mM, mixed 1:1 with 8 mg recombinant human stromelysin (mol. wt. 45-47 kDa, 2 Units; where 1 Unit produces 20 mmoles of Substance P 7-11 in 30 minutes) and incubated along with 0.5mM Substance P in a final volume of 0.125 mL for 30 minutes at 37xc2x0 C. The reaction is stopped by adding 10 mM EDTA and Substance P 7-11 is quantified on RP-8 HPLC. The IC50 for inhibition of stromelysin activity and Ki are calculated from control reaction without the inhibitor.
Stromelysin activity can also be determined using human aggrecan as a substrate. This assay allows the confirmation in-vitro that a compound can inhibit the action of stromelysin on its highly negatively-charged natural substrate, aggrecan (large aggregating proteoglycan). Within the cartilage, proteoglycan exists as an aggregate bound to hyaluronate. Human proteoglycan aggregated to hyaluronate is used as an enzyme substrate. The assay is set up in 96-well microtiter plates allowing rapid evaluation of compounds. The assay has three major steps:
1) Plates are coated with hyaluronate (human umbilical chord, 400 ug/mL), blocked with BSA (5 mg/mL), and then proteoglycan (human articular cartilage D1xe2x88x92chondroitinase ABC digested, 2 mg/mL) is bound to the hyaluronate. Plates are washed between each step.
2) Buffers+inhibitor (1 to 5,000 nM)+recombinant human stromelysin (1-3 Units/well) are added to wells. The plates are sealed with tape and incubated overnight at 37xc2x0 C. The plates are then washed.
3) A primary (3B3) antibody (mouse IgM, 1:10,000) is used to detect remaining fragments. A secondary antibody, peroxididase-linked anti-IgM, is bound to the primary antibody. OPD is then added as a substrate for the peroxidase and the reaction is stopped with sulfuric acid. The IC50 for inhibition of stromelysin activity is graphically derived and Ki is calculated.
Collagenase-1 inhibitory activity is determined as follows: ninety six-well, flat-bottom microtiter plates are first coated with bovine type I collagen (35 ug/well) over a two-day period at 30xc2x0 C. using a humidified and then dry atmosphere; plates are rinsed, air dried for 3-4 hours, sealed with Saran wrap and stored in a refrigerator. Human recombinant fibroblast collagenase and a test compound (or buffer) are added to wells (total volume=0.1 mL) and plates are incubated for 2 hours at 35xc2x0 C. under humidified conditions; the amount of coliagenase used per well is that causing approximately 80% of maximal digestion of collagen. The incubation media are removed from the wells, which are then rinsed with buffer, followed by water. Coomasie blue stain is added to the wells for 25 minutes, removed, and wells are again rinsed with water. Sodium dodecyl sulfate (20% in 50% dimethylformamide in water) is added to solubilize the remaining stained collagen and the optical density at 570 nM wave length is measured. The decrease in optical density due to collagenase (from that of collagen without enzyme) is compared to the decrease in optical density due to the enzyme in the presence of test compound, and percent inhibition of enzyme activity is calculated. IC50""s are determined from a range of concentrations of inhibitors (4-5 concentrations, each tested in triplicate), and Ki values are calculated.
Collagenase-3 inhibitory activity is determined as follows: One nM stock solutions of substrate (MCA-Pro-Leu-Gly-Dpa-Ala-Arg-NH2, J. Biol. Chem. 271, 1544-1550, 1996) and 10 nM stock solution of inhibitor are prepared in DMSO. They are diluted with assay buffer (20 nM tris at pH 7.5 containing 10 mM CaCl2, 0.002% sodium azide) as needed. Recombinant pro-collagenase-3 is activated with 1 mM APMA, and stored in the assay buffer after extensive dialysis in the assay buffer. Recombinant enzyme solution (0.05 mL, 1.3 nM) is mixed with 0.05 mL of inhibitor solution at various concentrations for 10 minutes at room temperature. Then 0.025 mL of 8 xcexcM substrate solution is added and fluorescence (xcexex=325; xcexem=405) is continuously measured at room temperature. The percent inhibition of collagenase-3 activity is determined from the effect of inhibitor at various concentrations on the change in fluorescence; the IC50 is determined graphically.
The effect of compounds of the invention in-vivo can be determined in rabbits. Typically, four rabbits are dosed orally with a compound up to four hours before being injected intra-articularly in both knees (N=8) with 40 Units of recombinant human stromelysin dissolved in 20 mM Tris, 10 mM CaCl2, and 0.15 M NaCl at pH 7.5. Two hours later the rabbits are sacrificed, synovial lavage is collected, and keratan sulfate (KS) and sulfated glycosaminoglycan (S-GAG) fragments released into the joint are quantitated. Keratan sulfate is measured by an inhibition ELISA using the method of Thonar (Thonar, E. J.-M.A., Lenz, M. E., Klinsworth, G. K., Caterson, B., Pachman, L. M., Glickman, P., Katz, R., Huff, J., Keuttner, K. E. Quantitation of keratan sulfate in blood as a marker of cartilage catabolism, Arthr. Rheum. 28, 1367-1376 (1985)). Sulfated glycosaminoglycans are measured by first digesting the synovial lavage with streptomyces hyaluronidase and then measuring DMB dye binding using the method of Goldberg (Goldberg, R. L. and Kolibas, L. An improved method for determining proteoglycan synthesized by chondrocytes in culture. Connect. Tiss. Res. 24, 265-275 (1990)). For an i.v. study, a compound is solubilized in 1 mL of PEG-400, and for a p.o. study, a compound is administered in 5 mL of fortified corn starch per kilogram of body weight.
The effect in protecting against cartilage degradation in arthritic disorders can be determined e.g. in a surgical model of osteoarthritis described in Arthritis and Rheumatism, Vol. 26, 875-886 (1983).
The effect on ulcerations, e.g. ocular ulcerations, can be determined in the rabbit by measuring the reduction of corneal ulceration following an alkali burn to the cornea.
Macrophage metalloelastase (MME) inhibitory activity can be determined by measuring the inhibition of the degradation of [3H]-elastin by truncated recombinant mouse macrophage metalloelastase as follows:
About 2 ng of recombinant truncated mouse macrophage metalloelastase (FASEB Journal Vol. 8, A151, 1994), purified by Q-Sepharose column chromatography is incubated with test compounds at the desired concentrations in the presence of 5 nM CaCl2, 400 nM NaCl, [3H]elastin (60,000 cpm/tube), and 20 mM Tris, pH 8.0, at 37xc2x0 C. overnight. The samples are spun in a microfuge centrifuge at 12,000 rpm for 15 minutes. An aliquot of the supernatant is counted in a scintillation counter to quantitate degraded [3H]elastin. IC50""s are determined from a range of concentrations of the test compounds and the percent inhibition of enzyme activity obtained.
The effect of the compounds of the invention for the treatment of emphysema can be determined in animal models described in American Review of Respiratory Disease 117, 1109 (1978).
The antitumor effect of the compounds of the invention can be determined e.g. by measuring the growth of human tumors implanted subcutaneously in Balb/c nude treated mice according to methodology well-known in the art in comparison to placebo treated mice. IIIustrative tumors are e.g. estrogen dependent human breast carcinoma BT20 and MCF7, human bladder carcinoma T24, human colon carcinoma Colo 205, human lung adenocarcinoma A549 and human ovarian carcinoma NIH-OVCAR3. 
The effect on tumor angiogenesis can be determined e.g. in rats implanted with Walker 256 carcinoma in pellets to stimulate angiogenesis from vessels of the limbus, as described by Galardy et al, Cancer Res. 54, 4715 (1994).
Gelatinase A and MT1-MMP inhibitory activities can be determined as follows: Stock solutions of substrate (MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, Knight, C. G., Willenbrock, F., Murphy, G., A novel coumarin-labelled peptide for sensitive continous assays of the matrix metalloproteinases, FEBS lett., 296, 263-266, (1992)), are prepared in 100% DMSO at a concentration 1.0 mM. Stock solutions of inhibitors are prepared in 100% DMSO. The inhibitor is diluted into the assays from a solution in 100% DMSO, and controls substitute an equal volume of DMSO so that the final DMSO concentration from inhibitor and substrate dilutions in all assays is 6.0%. Assays are performed in assay buffer (150 mM NaCl, 10 mM CaCl2, 50 mM Tris-Cl pH7.5, 0.05% Brij-35) containing 6.0% DMSO once the substrate and inhibitor are diluted into it. The substrate concentration used in the assays is 10 xcexcM. The test is carried out at 37xc2x0 C. The fluorescence changes are monitored using an excitation wavelength of 320 nm and an emission wavelength of 340 nm. The reaction mixture is added in duplicate to appropriate wells of a 96 well microfluor plate. The reaction mixture is preincubated with the inhibitor for 30 min, the reaction is started by the addition of MMP enzyme and the fluorescence intensity is measured for 10 min. A time point that is on a linear part of the curve is chosen to determine the activity. The inhibition results are expressed as the inhibitor concentrations that produced 50% inhibition (IC50) of the activity in the control (non-inhibited) reaction.
The inhibition of tumor metastasis can be determined in two lung metastasis models: In the B16-F10 melanoma model metastasis is measured by counting the numbers of lung metastasized melanoma nodules produced by intravenously injected B16-F10 melanoma cells into BDF1 treated mice according to methodology well known in the art. In the HT1080 model and metastasis is quantified by measuring the fluorescence intensity of enhanced green fluorescent protein (EGFP) in the lung of Balb/c nude mice produced by the metastasized tumor from intravenously injected GFP-expressing human fibrosarcoma HT1080 cells. The inhibition is obtained by comparison of compound treated and placebo treated mice in both methods. In HT1080 model, EGFP expressing HT1080 cell is prepared by limiting dilution method in the presence of geneticin after transfecting the EGFP expression vector (pEGFP-C1) (CLONTECH Laboratories Inc., Palo Alto, Calif.). A suspension of cells (106 cells/0.1 mL of PBS) is injected intravenously in Balb/c nude mice. After administering test compounds and vehicle p.o. 3 weeks, tumor metastasized lung of mice are removed after sacrifice and homogenized. After centrifugation, the cells are washed 3 times with lysing reagent (150 mM NH4Cl, 0.1 mM EDTA-4 Na, 10 mM KHCO3, pH 7.4) to lysis red blood cells and 2 times with PBS. After centrifugation, EGFP is extracted from cells by 10% Triton in PBS and poured into the wells of 96-well multi plate. The fluorescence intensity is determined by use of a fluorescence plate reader at the excitation and emission wave length of 485 and 530 nm, respectively.
The effect of the compounds of the invention on atherosclerotic conditions can be evaluated using atherosclerotic plaques from cholesterol-fed rabbits which contain activated matrix metalloproteinases as described by Sukhova et al, Circulation 90, I 404 (1994). The inhibitory effect on matrix metalloproteinase enzyme activity in rabbit atherosclerotic plaques can be determined by in situ zymography, as described by Galis et al, J. Clin. Invest. 94, 2493 (1994), and is indicative of plaque rupture.
The effect on vascular aneurysms, e.g. the inhibition of aneurysm formation, can be determined in experimental models such as Apo-E transgenic mice and/or LDL receptor knockout mice.
The effect on restenosis and vascular remodeling can be evaluated in the rat ballooned carotid artery model.
The effect on demyelinating disorders of the nervous system, such as multiple sclerosis, can be evaluated by measuring the reversal of experimental autoimmune encephalomyelitis in mice, e.g. as described by Gijbels et al, J. Clin. Invest. 94, 2177 (1994).
The compounds of the invention are particularly useful in mammals as antiinflammatory agents for the treatment of e.g. osteoarthritis, rheumatoid arthritis, and as antitumor agents for the treatment and prevention of tumors growth, tumor metastasis, tumor invasion or progression and as antiatherosclerotic agents for the treatment and prevention of the rupture of atherosclerotic plaques.
The present invention also relates to methods of using the compounds of the invention and their pharmaceutically acceptable salts, or pharmaceutical compositions thereof, in mammals for inhibiting the matrix-degrading metalloproteinases, e.g. stromelysin, gelatinase, coliagenase and macrophage metalloelastase, for inhibiting tissue matrix degradation, and for the treatment of matrix-degrading metalloproteinase dependent conditions as described herein, e.g. inflammation, rheumatoid arthritis, osteoarthritis, also tumors (tumor growth, metastasis, progression or invasion), pulmonary disorders, and the like described herein. Tumors (carcinomas) include mammalian breast, lung, bladder, colon, prostate and ovarian cancer, and skin cancer, including melanoma and Kaposi""s sarcoma.